Turfgrass varieties having desirable looking turf when mowed infrequently

ABSTRACT

Turfgrass varieties with desirable looking turf when mowed infrequently are provided. Also provided is a method of producing a turfgrass plant having desirable looking turf when mowed infrequently. The turfgrass plants of the present invention display green leaf chlorophyll concentrations above 1.8 mg/g and a field insitu CM-1000 chlorophyll meter reading of 341.7 or higher, as well as high general turfgrass quality ratings. The turfgrass plants of the present invention retain a dark green color even when scalped back by infrequent mowing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. 371 ofInternational Application PCT/US2014/024563, filed Mar. 12, 2014,published in English, which claims the benefit of priority from U.S.provisional patent application Ser. No. 61/782,611, filed on Mar. 14,2013. The disclosure of each of the above-listed priority applicationsis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to turfgrass varieties having desirablelooking turf when mowed infrequently. All publications cited in thisapplication are herein incorporated by reference.

Turfgrass plays a major role in our daily life. Turfgrass, from abeautification standpoint, provides a canvas for landscaped areascontributing to aesthetic appeal and adding to economic value.Recreational facilities include an array of sports fields, golf courses,parks and lawns. Turfgrass also provides functional value including dustcontrol, erosion control, and glare reduction.

Use and appearance are prime considerations for turfgrass. To best servea particular function, the turf should be suitable for the use for whichit is intended and aesthetically appealing. It should also bewell-adapted to the environment where it will be planted. Based onclimatic adaptation, turfgrass species have been placed into fourcategories: adapted for cool humid regions, warm humid regions, coolarid regions, and warm arid regions. The major turfgrasses adapted tothe cool humid regions, and irrigated areas of the cool arid regions,are species of Agrostis, Poa, Festuca, and Lolium. In the warm humid andirrigated areas of the warm arid regions, the major adapted turfgrassesare species of Cynodon, Zoysia, Stenotaphrum, Eremochloa, Paspalum,Festuca, and Agropyron. In the non-irrigated warm arid regions, speciesof Buchloe and Bouteloua are adapted.

Kentucky bluegrass (Poa pratensis), also called smooth meadow grass,spear grass and June grass, is a perennial species of grass native toEurope, northern Asia and the mountains of Algeria and Morocco. Althoughthe species is spread over all of the cool, humid parts of the UnitedStates, Kentucky bluegrass is native only to portions of North America.Kentucky bluegrass forms a valuable pasture plant, characteristic ofwell-drained, fertile soil, and is a popular sod-forming grass that isused on golf courses, ski slopes, campsites, gardens and lawns. Kentuckybluegrass is also an important forage species for sheep and cattle. Thename Kentucky bluegrass derives from its flower heads, which are bluewhen the plant is allowed to grow to its natural height of two to threefeet.

Over 100 varieties of Kentucky bluegrass have been developed during thepast 25 years. Some varieties tolerate southern climates better thanothers, some have moderate shade tolerance, and some tolerate closermowing. Many of these grasses also differ in their degree ofsusceptibility to diseases. Kentucky bluegrass is distinguished fromCanada bluegrass (Poa compressus) by its darker green foliage, longerleaves, and pubescence at the bases of the leaves. Kentucky bluegrasscan also be compared to Annual Meadowgrass (Poa annua) and RoughMeadowgrass (Poa trivialis), which have a ligule that is silvery andpointed, whereas Kentucky bluegrass has a ligule that is extremely shortand square ended.

Kentucky bluegrass is often included in seed mixes that are used torevegetate roadbanks Kentucky bluegrass is a slow-growing plant thatestablishes in 2 to 3 years and forms a dense sod. Kentucky bluegrassgrows best on well-drained loams or clay loams rich in humus and onsoils with limestone parent material. Kentucky bluegrass needs largeamounts of nitrogen during active growth stages and has an optimal soilpH of between 5.8 and 8.2. Additionally, Kentucky bluegrass isintolerant of drought, excessive flooding high water tables, and poorlydrained soils, and is sometimes vulnerable to fungal infectionsincluding Fusarium, Helminthosporium, leaf spot, rust and powderymildew.

Kentucky bluegrass typically grows 18 to 24 inches tall and is readilyidentified by its boat-shaped leaf tip. Kentucky bluegrass spreads byrhizomes and tillers and forms a dense sod. New shoots (rhizomes andtillers) are produced primarily in the spring and late summer. Mostshoots produced in the spring remain vegetative, while shoots producedin late summer often terminate in an inflorescence the following spring.The lifetime of a Kentucky bluegrass shoot that terminates in aninflorescence ends soon after the seeds mature.

Because use and appearance are prime considerations for turfgrass, it isdesirable to have turfgrass varieties that can be less frequently mowed(defoliated) and still produce an attractive, green lawn turf.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

In one aspect of the present invention there is provided a method ofproducing a turfgrass plant having desirable looking turf when mowedinfrequently.

In one aspect of the invention there are provided turfgrass varietiesthat produce a desirable looking turf when mowed infrequently, such asmowed once every 4 weeks or more.

In one aspect of the present invention there are provided turfgrassvarieties that have chlorophyll concentrations above 1.8 mg/g.

In one aspect of the present invention there are provided turfgrassvarieties that have a CM-1000™ field chlorophyll reading of 341.7 orhigher resulting in a darker green color and less brown when mowed.

In another aspect of the present invention there are provided turfgrassvarieties that have low-growing characteristics.

In another aspect of the present invention there are provided turfgrassvarieties that have high general turfgrass quality ratings.

In a further aspect of the present invention there are provided Kentuckybluegrass plants of varieties ‘03-0582’, ‘03-0441’, and ‘99-2495’. Alsoprovided are seeds and progeny of these varieties.

According to the invention, there are provided novel turfgrass varietieshaving desirable looking turf when mowed infrequently, includingKentucky bluegrass varieties designated ‘03-0582’, ‘03-0441’, and‘99-2495’. This invention thus relates to the seeds of novel turfgrassvarieties, such as ‘03-0582’, ‘03-0441’, and ‘99-2495’, to the plants orpart(s) thereof of novel turfgrass varieties, such as Kentucky bluegrassvarieties ‘03-0582’, ‘03-0441’, and ‘99-2495’, to plants or part(s)thereof having all the phenotypic and morphological characteristics ofnovel turfgrass varieties, such as Kentucky bluegrass varieties‘03-0582’, ‘03-0441’, and ‘99-2495’. Plant parts of the Kentuckybluegrass varieties of the present invention are also provided.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of turfgrass varieties having desirable lookingturf when mowed infrequently, including Kentucky bluegrass varieties‘03-0582’, ‘03-0441’, and ‘99-2495’. The tissue culture will preferablybe capable of regenerating plants having the physiological andmorphological characteristics of the turfgrass of the present invention,such as Kentucky bluegrass varieties ‘03-0582’, ‘03-0441’, and‘99-2495’. Preferably, the cells of such tissue culture will be embryos,meristematic cells, seeds, callus, pollen, leaves, anthers, pistils,roots, root tips, pods, flowers and stems. Protoplasts produced fromsuch tissue culture are also included in the present invention. Theturfgrass plants regenerated from the tissue culture are also part ofthe invention.

Also included in the invention are methods for producing a turfgrassplant produced by crossing turfgrass varieties having desirable lookingturf when mowed infrequently, such as Kentucky bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’ with itself or another turfgrass orKentucky bluegrass variety. When crossed with itself, i.e., when crossedwith another Kentucky bluegrass variety ‘03-0582’, ‘03-0441’, or‘99-2495’ plant or self-pollinated, Kentucky bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’ will be conserved. When crossed withanother, different bluegrass plant, an F₁ hybrid seed is produced. F₁hybrid seeds and plants produced by growing said hybrid seeds areincluded in the present invention. A method for producing an F₁ hybridseed comprising crossing a turfgrass plant of the present inventionhaving desirable looking turf when mowed infrequently, such as Kentuckybluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ plant with adifferent bluegrass plant and harvesting the resultant hybrid bluegrassseed are also part of the invention. The hybrid bluegrass seed producedby the method comprising crossing a turfgrass plant having desirablelooking turf when mowed infrequently, such as Kentucky bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’ plant with a different bluegrassplant and harvesting the resultant hybrid bluegrass seed, are includedin the invention, as are the hybrid bluegrass plant or part(s) thereof,and seeds produced by growing said hybrid bluegrass seed.

In another aspect, the present invention provides transformed turfgrassvarieties having desirable looking turf when mowed infrequently,including ‘03-0582’, ‘03-0441’, or ‘99-2495’ Kentucky bluegrass varietyplants or part(s) thereof that have been transformed so that its geneticmaterial contains one or more transgenes, preferably operably linked toone or more regulatory elements. Also, the invention provides methodsfor producing a turfgrass plant having desirable looking turf when mowedinfrequently containing in its genetic material one or more transgenes,preferably operably linked to one or more regulatory elements, bycrossing transformed turfgrass plants, such as ‘03-0582’, ‘03-0441’, or‘99-2495’ Kentucky bluegrass variety plants with either a second plantof another turfgrass or bluegrass variety, or a non-transformed‘03-0582’, ‘03-0441’, or ‘99-2495’ Kentucky bluegrass variety, so thatthe genetic material of the progeny that results from the cross containsthe transgene(s), preferably operably linked to one or more regulatoryelements. The invention also provides methods for producing a turfgrassplant having desirable looking turf when mowed infrequently thatcontains in its genetic material one or more transgene(s), wherein themethod comprises crossing the turfgrass variety, such as ‘03-0582’,‘03-0441’, or ‘99-2495’ with a second turfgrass or bluegrass variety ofanother bluegrass variety which contains one or more transgene(s)operably linked to one or more regulatory element(s) so that the geneticmaterial of the progeny that results from the cross contains thetransgene(s) operably linked to one or more regulatory element(s).Transgenic turfgrass cultivars, or part(s) thereof produced by themethods are in the scope of the present invention.

More specifically, the invention comprises methods for producing a malesterile turfgrass plant, an herbicide resistant turfgrass plant, aninsect resistant turfgrass plant, a disease resistant turfgrass plant, awater stress tolerant turfgrass plant, a heat stress tolerant turfgrassplant, and a turfgrass plant with improved shelf-life. Said methodscomprise transforming a turfgrass variety of the present invention, suchas ‘03-0582’, ‘03-0441’, or ‘99-2495’ plant with a nucleic acid moleculethat confers male sterility, herbicide resistance, insect resistance,disease resistance, water stress tolerance, heat stress tolerance, orimproved shelf life, respectively. The transformed turfgrass plants, orpart(s) thereof, obtained from the provided methods, including a malesterile turfgrass plant, an herbicide resistant turfgrass plant, aninsect resistant turfgrass plant, a disease resistant turfgrass plant, aturfgrass plant tolerant to water stress, a turfgrass plant tolerant toheat stress or a turfgrass plant with improved shelf-life are includedin the present invention. For the present invention and the skilledartisan, disease is understood to be fungal diseases, viral diseases,bacterial diseases or other plant pathogenic diseases and a diseaseresistant plant will encompass a plant resistant to fungal, viral,bacterial and other plant pathogens.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into turfgrass varieties havingdesirable looking turf when mowed infrequently, such as bluegrassvarieties ‘03-0582’, ‘03-0441’, or ‘99-2495’ and plants obtained fromsuch methods. The desired trait(s) may be, but not exclusively, a singlegene, preferably a dominant but also a recessive allele. Preferably, thetransferred gene or genes will confer such traits as male sterility,herbicide resistance, insect resistance, resistance to bacterial,fungal, or viral disease, increased leaf number, improved shelf-life,and tolerance to water stress or heat stress. The gene or genes may benaturally occurring gene(s) or transgene(s) introduced through geneticengineering techniques. The method for introducing the desired trait(s)is preferably a backcrossing process making use of a series ofbackcrosses to turfgrass plants having desirable looking turf when mowedinfrequently, such as bluegrass variety ‘03-0582’, ‘03-0441’, or‘99-2495’ during which the desired trait(s) is maintained by selection.

In a preferred embodiment, the present invention provides methods forincreasing and producing turfgrass varieties having desirable lookingturf when mowed infrequently, such as bluegrass varieties ‘03-0582’,‘03-0441’, or ‘99-2495’ seed, whether by crossing a first parentbluegrass variety plant with a second parent bluegrass variety plant andharvesting the resultant bluegrass seed, wherein both said first andsecond parent bluegrass variety plant are the bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’ or by planting a bluegrass seed ofthe bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’, growing abluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ plant from saidseed, controlling a self pollination of the plant where the pollenproduced by a grown bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’plant pollinates the ovules produced by the very same bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’ grown plant, and harvesting theresultant seed.

The invention further provides methods for developing turfgrass andKentucky bluegrass cultivars having desirable looking turf when mowedinfrequently in a turfgrass breeding program using plant breedingtechniques including recurrent selection, backcrossing, pedigreebreeding, molecular markers (Isozyme Electrophoresis, RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs). Amplified Fragment Length Polymorphisms (AFLPs), andSimple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, etc.) enhanced selection, genetic marker enhancedselection, and transformation. Seeds, turfgrass plants, and part(s)thereof produced by such breeding methods are also part of theinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of regression analysis that was performed tocorrelate visual turfgrass quality ratings with Field Scout CM 1000™scan results for test plots mowed three times per year in Idaho.Regression analysis indicated that a visual quality estimate of 5corresponds to a CM 1000™ meter reading of 341.7, with an unexpectedlyhigh correlation co-efficient of 0.86, and an R² value of 0.73, whichwas significant at the 0.0001 level. In FIG. 1, circles representindividual observations, a solid line indicates regression fit to thedata, and dashed lines indicate 95% confidence intervals.

DETAILED DESCRIPTION OF THE INVENTION

In the description and examples that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. If no definition is provided,all other technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to whichthe invention belongs.

Acceptable level of apomixes. Refers to 80% or higher level of apomixes.

Adequate production of seed heads. Refers to 15 grams or more of cleanseed recovered from one individual spaced plant.

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Alter. Alter refers to the utilization of up-regulation,down-regulation, or gene silencing.

Apomictic. As used herein, “apomictic” describes a plant that reproducesusing apomixis

Apomixis. Asexual reproduction in organisms that are also able toreproduce sexually, in which embryos are formed without fertilization orthe creation of specialized reproductive cells.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

Characteristics different from the maternal parental line. As usedherein, refers to characteristics that are different from the maternalparental line, including but not limited to color and width of theleaves prior to seedhead expression and a different date when theseedhead emerges from the sheath of the turfgrass.

Chlorophyll concentration. The milligrams per gram of chlorophyllcontained in plant tissue weight. Also referred to as “chlorophyllcontent”.

Chlorophyll meter reading. The digital readout of the CM 1000™chlorophyll field scanner.

Cisgenesis. The genetic modification of a recipient plant with a naturalgene from a sexually compatible plant. Such a gene includes its intronsand is flanked by it native promoter and terminator in the normal senseorientation. Cisgenic plants can harbor one or more cisgenes, but theydo not contain any trangenes.

Cisgenic plant. A plant that contains no foreign genes.

Commercial perennial bluegrass. A commercial perennial bluegrass is onewhich has been sold commercially.

Cotyledon. A cotyledon is a seed leaf.

Crossbreeding. As used herein, “crossbreeding” refers to the act ofmating (crossing) individuals of different species or varieties ofplants to produce hybrids.

Crown. The crown in grass is the area at which top growth and rootgrowth originate.

Culm. The culm is the main aerial shoot to which leaves andinflorescences are attached. The culm is a rounded or slightly flattenedstem with one or more solid joints known as nodes. The leaves areattached at the nodes and if the stem is not simple but branched,branches arise only at nodes. Roots may also develop from a node wherethe node comes into contact with the ground (as in decumbent andprostrate stems).

Desirable looking turf As used herein, “desirable looking turf” isquantified by an increased chlorophyll concentration of greater than 1.8mg/g indicating the turf retention of green color even when scalped back(severely defoliated) by infrequent mowing. The chlorophyllconcentration is additionally quantified by a CM-1000™ field chlorophyllreading of 341.7 or higher. “Turf” refers to any type of turfgrass,including bluegrass.

Embryo. The embryo is the small plant contained within a mature seed.

Endophyte. The term endophyte is applied to fungi which live withinplant tissues for all or part of their lifecycle and cause no apparentinfections.

Famigenic plant. A transformed plant developed by transferring at leastsome DNA from one plant to a sexually incompatible plant that belongs tothe same family.

Field hybridization nursery. A spaced plant field breeding nurseryhaving access to plants that grew from one seed, wherein a mother plantis surrounded by clones of itself (selfing) or a mother plant issurrounded by clones that are different than the mother plant(intraspecific hybridization).

Field Scout CM 1000™ chlorophyll meter. A “point-and-shoot” device toinstantly measure relative chlorophyll content. The CM 1000™ senseslight at wavelengths of 700 nm and 840 nm to estimate the quantity ofchlorophyll in leaves. The ambient and reflected light at eachwavelength is measured. Made by Spectrum Technologies, Inc., Plainfield,Ill.

Gene. As used herein, “gene” refers to a DNA segment that contributes tophenotype/function, including associated regulatory elements such aspromoters. A gene can be introduced into a genome of a species, whetherfrom a different species or from the same species, using transformationor various breeding methods.

Gene silencing. The interruption or suppression of the expression of agene at the level of transcription or translation.

Genotype. Refers to the genetic constitution of a cell or organism.

Grass flower or inflorescence. Flowers of grasses are borne in aninflorescence or flower head which terminates the culm and otherbranches of the stem. Smaller units of the inflorescence are calledspikelets and these are arranged on one or more branches in a widevariety of different ways to which the standard terminology forinflorescences can be applied, but using the spikelet instead of theindividual flower.

Growing season. As used herein, “growing season” refers to the time ofyear during which turfgrass is actively growing, which is typicallyspring through fall in the U.S. and Europe.

Hybrid. Heterozygous offspring of two parents that differ in one or moreinheritable characteristics.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Intragenic plant. A transformed plant that only contains geneticelements derived from within the sexual compatibility group.

Intergenic DNA. Any of the DNA in between gene-coding DNA, includinguntranslated regions, 5′ and 3′ flanking regions, introns,non-functional pseudogenes, and non-functional repetitive sequences.This DNA may or may not encode regulatory functions.

Internode. The internodes act as spacers that distance one node fromanother.

Intercalary meristem. Intercalary meristem is a meristem at the base ofthe internode in monocot stems (particularly grass stems).

Lawn. A plot of grass, usually tended or mowed, such as one near ahouse, on an estate, in a yard, garden or park, or a golf course, or anyother such area covered with grass.

Linkage. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

Linkage disequilibrium. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

Mature sod. Means sod that is 8 to 14 months old after seeding, whereinsod is mature at 8 to 11 months old after a fall seeding and sod ismature at 12 to 14 months old after a spring seeding.

Mowed infrequently. As used herein, “mowed infrequently” or“infrequently mowed” refers to grass that has been subjected to mowingwith a conventional lawnmower once every four weeks or longer, such asonce every 30, 32, 34, 36, 38, 39, 40 or more days, or any integer orfraction thereof.

Node. A node in a grass stem is a solid point at which the intercalarymeristem is located. The node also contains the bud that is capable ofproducing a new shoot. The terminal node contains the bud that producesthe inflorescence.

Pedigree distance. Pedigree distance refers to the relationship amonggenerations based on their ancestral links as evidenced in pedigrees.Pedigree distance may be measured by the distance of the pedigree from agiven starting point in the ancestry.

Percent identity. Percent identity as used herein refers to thecomparison of the homozygous alleles of two perennial bluegrassvarieties. Percent identity is determined by comparing a statisticallysignificant number of the homozygous alleles of two developed varieties.For example, a percent identity of 90% between perennial bluegrassvariety 1 and perennial bluegrass variety 2 means that the two varietieshave the same allele at 90% of their loci.

Percent similarity. Percent similarity as used herein refers to thecomparison of the homozygous alleles of one perennial bluegrass varietywith another bluegrass plant, and if the homozygous allele of the firstbluegrass matches at least one of the alleles from the other plant thenthey are scored as similar. Percent similarity is determined bycomparing a statistically significant number of loci and recording thenumber of loci with similar alleles as a percentage. A percentsimilarity of 90% between the first bluegrass and another plant meansthat the first bluegrass matches at least one of the alleles of theother plant at 90% of the loci.

Plant. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant from which seed, rootsor leaves have been removed. Seed or embryo that will produce the plantis also considered to be the plant.

Plant height. The length of the grass leaf blade measured from therhizome to the tip of the blade.

Plant parts. As used herein, the term “plant parts” (or a perennialbluegrass plant, or a part thereof) includes protoplasts, leaves, stems,roots, root tips, anthers, pistils, seed, embryo, pollen, ovules,cotyledon, hypocotyl, flower, shoot, tissue, petiole, cells,meristematic cells and the like.

Polynucleotide. A polymeric compound, usually DNA or RNA, consisting ofa number of nucleotides.

Primary tillers. Primary tillers are shoots arising at the crown.

Progeny. As used herein, includes an F₁ turfgrass plant produced fromthe cross of two turfgrass plants where at least one plant includes aturfgrass plant of the present invention and progeny further includes,but is not limited to, subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, andF₁₀ generational crosses with the recurrent parental line. As usedherein, progeny also refers to plants produced by selfing a turfgrassplant produced by the present invention.

Promoter. A segment of DNA usually occurring upstream from a gene codingregion and acting as a controlling element in the expression of thatgene.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Rhizome. A rhizome is a modified stem that grows underground. Rhizomesare jointed (thus distinguishable from roots) with bladeless leaves(scales) arising from the joints Rhizomes enable a grass plant to spreadhorizontally as new culms develop vertically from the joints. Thus,grasses with extensive rhizome development will form a turf rather thandistinct tufts or bunches.

Scalped back (severely defoliated). Refers to mowing of turfgrasswherein greater than one third of the leaf area is removed.

Secondary tillers. Secondary tillers are tillers arising as branches ofthe primary tillers.

Seedhead. The flowering (reproductive) part of the grass plant.

Seedhead expression. Refers to the emergence, full expression andmaturation of the seedhead.

Selfing. Pollinating a plant and hybridizing it with its own pollen orpollen from a clone of that plant.

Short, basal growth of leaves. Refers to a preponderance of green leafmaterial in the 0 to 4 inch zone above the soil surface.

Single Gene Converted (Conversion). Single gene converted (conversion)plants refers to plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique or via genetic engineering.

Sod. A section of grass-covered surface soil held together by mattedroots; turf

Stolon. A stolon is a stem that creeps across the surface of the ground,and is really a basal branch of the culm that will develop roots andshoots from some or all of its nodes. Like a rhizome, a stolon resultsin a spreading or turf forming grass plant.

Tensile strength. Means the amount of force in pounds required to tear apiece of sod in two. Tensile strength is determined with a mechanicalsod stretcher coupled to a device to measure force in pounds. Tensilestrength, tear point and sod strength are used interchangeably.

Tiller. A tiller is another name for a grass stem.

Tiller length. Tiller length is measured in centimeters from the lowestnode to the last node subtending the green foliage.

Transgene. A gene that is transferred from an organism of one species toan organism of another species by genetic engineering.

Variant. As used herein, refers to offspring that occur in a naturallyapomictic species by means of hybridization of pollen and ovule,resulting in offspring with some but not all characteristics of thematernal parent plant.

Variety. A taxonomic subdivision of a species consisting of naturallyoccurring or selectively bred populations or individuals that differfrom the remainder of the species in certain minor characteristics.

Vernalization. Vernalization induces plants to begin the reproductivecycle after exposure to cold temperatures and short day length. Theamount of cold exposure and short day lengths required varies with thespecies.

Xenogenic modification. Introduction of powerful new traits that mightoutperform native traits by transforming plants with synthetic genes.

The following detailed description is of the currently contemplatedmodes of carrying out the invention. The description is not to be takenin a limiting sense, but is made merely for the purpose of illustratingthe general principles of the invention, since the scope of theinvention is best defined by the appended claims.

Broadly, the present invention is directed toward turfgrass varieties,including both plants and seeds, having desirable looking turf whenmowed infrequently. The turfgrass varieties of the present inventionwere developed to retain a dark green color even when scalped back byinfrequent mowing. Part of this tolerance traces to the method ofbreeding, which stresses low growing plants, with foliage tight to theground. However, there are also other traits that are less quantitativethat convey infrequent mowing tolerance, such as the physiologicalability to withstand defoliation and recover without discoloration orobjectionable stunting. In essence, these plants do not just growvertically slower than other varieties, they also possess growthcharacteristics that allow them to endure on a infrequently mowed lawnwhile maintaining a desirable looking turf.

In the field hybridization nursery, individual spaced plants areidentified with promising characteristics such as the short growth ofleaves (defined as a preponderance of green leaf material in the 0 to 4inch zone above the soil surface), adequate seed heads (defined as 15 gor more of clean seed recovered from one individual spaced plant), andacceptable apomixes (defined as 80% or higher). These promising plantsare individually hand harvested and cleaned of chaff and seed areplanted in replicated turf trials, subject to mowing once every 4 weeksor longer. Each plot in the experiment is evaluated monthly during thegrowing season, using a visual rating scale of 1 to 9, where 9 is highlydesirable turf and 5 is minimal acceptable quality, and 1 is totallybrown or dead. During one or more evaluation dates, the chlorophyllmeter is used to evaluate plots to impartially differentiate greentissue from lifeless brown tissue created from the scalping process.

Most turfgrass managers practice the “one third rule” for clipping lawnsand golf courses. The “one third rule” states that a maximum of onethird of the leaf area should be removed during any one mowing.Deviating from this “one third rule” could lead to an unacceptablequantity of the plant's photosynthetic surface being removed. As aresult, the plant goes into a shock mode and sacrifices leaves inexchange for survival. Plant varieties that grow vertically very tall orextremely fast are more subject to damage from defoliation. The visualresult is that the plant rapidly turns brown within 24 hours and maytake days or weeks to recover. An additional result of this shock is theloss of shoot density of the plant, defined as the number of livevegetative shoots per square centimeter of ground surface. The presentinvention described herein deals with plants that do not exhibit thisbrowning shock when they are subject to infrequent mowing.

The present invention provides a method of producing a turfgrass planthaving desirable-looking turf when mowed infrequently, said methodcomprises selfing (pollinating a plant and hybridizing it with its ownpollen) for one or more generations to create a plant with short growthof leaves (defined as a preponderance of green leaf material in the 0 to4 inch zone above the soil surface), adequate seed heads (defined as 15g or more of clean seed recovered from one individual spaced plant), andacceptable apomixes (defined as 80% or higher). The hybridization cantake place in a confined greenhouse using pollen restricting bags, ortake place in the field surrounded by clones of the same genotype, orany combination of these two methods. The progeny of said hybridizationsare then field screened for approximately 15 months to identify plantswith characteristics different than the maternal parent line; suchdifferences are most commonly identified during the vegetative orpre-seedhead phase, but also may be identified during seedheadexpression. The method of the present invention unexpectedly producedturfgrass plants having very basal growth habit, with leaves growingclose to the ground rather than growing tall, which havedesirable-looking turf when mowed infrequently.

It should be understood, of course, that the foregoing relates toexemplary embodiments of the invention and that modifications may bemade without departing from the spirit and scope of the invention as setforth in the following claims.

FURTHER EMBODIMENTS OF THE INVENTION

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). Popular selection methods commonly include populationformation by hybridization, genomic selection, marker assistedselection, recurrent selection, mutation breeding, single-seed descent,bulk selection, pedigree selection, modified pedigree selection, andmass selection.

Breeding Methods

The following describes breeding methods that may be used with theturfgrass varieties of the present invention having desirable lookingturf when mowed infrequently, such as bluegrass varieties ‘03-0582’,‘03-0441’, or ‘99-2495’ in the development of further bluegrass plants.One such embodiment is a method for developing a cultivar ‘03-0582’,‘03-0441’, or ‘99-2495’ progeny bluegrass plant in a bluegrass plantbreeding program comprising: obtaining the bluegrass plant, or a partthereof, of cultivar ‘03-0582’, ‘03-0441’, or ‘99-2495’ utilizing saidplant or plant part as a source of breeding material and selecting abluegrass cultivar ‘03-0582’, ‘03-0441’, or ‘99-2495’ progeny plant withmolecular markers in common with variety ‘03-0582’, ‘03-0441’, or‘99-2495’ and/or with morphological and/or physiological characteristicsdescribed herein.

Another method involves producing a population of turfgrass varieties ofthe present invention having desirable looking turf when mowedinfrequently, such as bluegrass varieties ‘03-0582’, ‘03-0441’, or‘99-2495’ progeny bluegrass plants, comprising crossing cultivar‘03-0582’, ‘03-0441’, or ‘99-2495’ with another bluegrass plant, therebyproducing a population of bluegrass plants, which, on average, derive50% of their alleles from bluegrass variety ‘03-0582’, ‘03-0441’, or‘99-2495’. A plant of this population may be selected and repeatedlyselfed or sibbed with a bluegrass cultivar resulting from thesesuccessive filial generations. In some embodiments, the bluegrasscultivar produced by this method and that has obtained at least 50% ofits alleles from bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the methods and variety describedherein includes turfgrass plants having desirable looking turf whenmowed infrequently, such as bluegrass cultivar ‘03-0582’, ‘03-0441’, or‘99-2495’ progeny bluegrass plants comprising a combination of at leasttwo cultivar ‘03-0582’, ‘03-0441’, or ‘99-2495’ traits or the cultivar‘03-0582’, ‘03-0441’, or ‘99-2495’ combination of traits listed in theExamples, so that said progeny bluegrass plant is not significantlydifferent for said traits than bluegrass variety ‘03-0582’, ‘03-0441’,or ‘99-2495’ as determined at the 5% significance level when grown inthe same environmental conditions. Using techniques described herein,molecular markers may be used to identify said progeny plant as abluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ progeny plant. Meantrait values may be used to determine whether trait differences aresignificant, and preferably the traits are measured on plants grownunder the same environmental conditions. Once such a variety isdeveloped its value is substantial since it is important to advance thegermplasm base as a whole in order to maintain or improve traits such asyield, disease resistance, pest resistance, and plant performance inextreme environmental conditions.

Progeny of turfgrass plants of the present invention having desirablelooking turf when mowed infrequently, such as bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’ may also be characterized throughtheir filial relationship with bluegrass variety ‘03-0582’, ‘03-0441’,or ‘99-2495’, as for example, being within a certain number of breedingcrosses of bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’. Abreeding cross is a cross made to introduce new genetics into theprogeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ and itsprogeny. For example, progeny produced by the methods described hereinmay be within 1, 2, 3, 4 or 5 breeding crosses of bluegrass variety‘03-0582’, ‘03-0441’, or ‘99-2495’.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Turfgrass having desirable looking turfwhen mowed infrequently, such as ‘03-0582’, ‘03-0441’, or ‘99-2495’ issuitable for use in a recurrent selection program. The method entailsindividual plants cross pollinating with each other to form progeny. Theprogeny are grown and the superior progeny selected by any number ofselection methods, which include individual plant, half-sib progeny,full-sib progeny and selfed progeny. The selected progeny are crosspollinated with each other to form progeny for another population. Thispopulation is planted and again superior plants are selected to crosspollinate with each other. Recurrent selection is a cyclical process andtherefore can be repeated as many times as desired. The objective ofrecurrent selection is to improve the traits of a population. Theimproved population can then be used as a source of breeding material toobtain new varieties for commercial or breeding use, including theproduction of a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedvarieties. The number of parental plant varieties, populations, wildaccessions, ecotypes, etc., that are used to generate a synthetic canvary from as little as 10 to as much as 500. Typically, about 100 to 300varieties, populations, etc., are used a parents for the syntheticvariety. Seed from the parental seed production plot of a syntheticvariety can be sold to the farmer. Alternatively, seed from the parentalseed production plot can subsequently undergo one or two generations ofmultiplication, depending on the amount of seed produced in the parentalplot and the demand for seed.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self pollination, directed pollinationcould be used as part of the breeding program.

Mutation breeding is another method of introducing new traits intoturfgrass plants having desirable looking turf when mowed infrequently,such as bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’. Mutationsthat occur spontaneously or are artificially induced can be usefulsources of variability for a plant breeder. The goal of artificialmutagenesis is to increase the rate of mutation for a desiredcharacteristic. Mutation rates can be increased by many different meansincluding temperature, long-term seed storage, tissue cultureconditions, radiation; such as X-rays, Gamma rays (e.g. cobalt 60 orcesium 137), neutrons, (product of nuclear fission by uranium 235 in anatomic reactor), Beta radiation (emitted from radioisotopes such asphosphorus 32 or carbon 14), or ultraviolet radiation (such as from 2500to 2900 nm), or chemical mutagens (such as base analogues(5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones),azide, hydroxylamine, nitrous acid, or acridines. Once a desired traitis observed through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Fehr, 1993. Principles of CultivarDevelopment, Macmillan Publishing Company. In addition, mutationscreated in other bluegrass plants may be used to produce a backcrossconversion of bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ thatcomprises such mutation.

Breeding with Molecular Markers

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing bluegrass variety ‘03-0582’, ‘03-0441’, or‘99-2495’.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (O'Brien, S. J., (ed.) 1993.Genetic Maps: Locus Maps of Complex Genomes. Cold Spring HarborLaboratory Press. Cold Spring Harbor, N.Y.), developed a moleculargenetic linkage map that consisted of 25 linkage groups with about 365RFLP, 11 RAPD (random amplified polymorphic DNA), three classicalmarkers, and four isozyme loci. See also, Shoemaker R. C. 1994. “RFLPMap of Soybean” p 299-309 In R. L. Phillips and I. K. Vasil (ed.)DNA-Based Markers in Plants. Kluwer Academic Press Dordrecht, theNetherlands. In switchgrass, Missaoui also described RFLP markers(Missaoui et al., 2006, “Molecular markers for the classification ofswitchgrass (Panicum virgatum L.) germplasm and to assess geneticdiversity in three synthetic switchgrass populations” Genetic Resourcesand Crop Evolution 53:1291-1302).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. For exampleDiwan and Cregan, described a highly polymorphic microsatellite loci insoybean with as many as 26 alleles. (Diwan, N., and P. B. Cregan. 1997“Automated sizing of fluorescent-labeled simple sequence repeat (SSR)markers to assay genetic variation in soybean”. Theor. Appl. Genet.95:220-225). Single Nucleotide Polymorphisms (SNPs) may also be used toidentify the unique genetic composition of ‘03-0582’, ‘03-0441’, or‘99-2495’ and progeny varieties retaining that unique geneticcomposition. Various molecular marker techniques may be used incombination to enhance overall resolution.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Gene Conversions

When the term “turfgrass plant” or “bluegrass plant” is used in thecontext of the methods and varieties described herein, this alsoincludes any gene conversions of that variety. The term gene convertedplant as used herein refers to those turfgrass plants which aredeveloped by a plant breeding technique called backcrossing whereinessentially all of the desired morphological and physiologicalcharacteristics of a variety are recovered in addition to the one ormore genes transferred into the variety via the backcrossing technique.Backcrossing methods can be used with the methods and variety describedherein to improve or introduce one or more characteristics into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, i.e.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to the recurrentparent. The parental turfgrass plant that contributes the gene(s) forthe desired characteristic is termed the nonrecurrent or donor parent.This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and therefore does not recur. Theparental turfgrass plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, Principles of Cultivar Development pp. 261-286(1987)). In a typical backcross protocol, the original variety ofinterest (recurrent parent) is crossed to a second variety (nonrecurrentparent) that carries the gene(s) of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a turfgrass plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the transferred gene(s) from thenonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more traits or characteristics in theoriginal variety. To accomplish this, one or more genes of the recurrentvariety is/are modified or substituted with the desired gene(s) from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic(s) or trait(s) being altered to determinean appropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic(s) has been successfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. Traits may or may not be transgenic; examplesof these traits include but are not limited to, male sterility,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, male fertility, enhanced nutritionalquality, modified oil content, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these traits are described in U.S. Pat. Nos.5,959,185; 5,973,234 and 5,977,445; the disclosures of which arespecifically hereby incorporated by reference for this purpose.

Introduction of a New Trait or Locus into Turfgrass Plants HavingDesirable Looking Turf when Mowed Infrequently, Such as ‘03-0582’,‘03-0441’, or ‘99-2495’

Turfgrass plants of the present invention having desirable looking turfwhen mowed infrequently, such as variety ‘03-0582’, ‘03-0441’, or‘99-2495’ represent a new base genetic variety into which a new locus ortrait may be introgressed. Direct transformation and backcrossingrepresent two important methods that can be used to accomplish such anintrogression. The term backcross conversion and locus conversion areused interchangeably to designate the product of a backcrossing program.

Backcross Conversions of Turfgrass Plants Having Desirable Looking Turfwhen Mowed Infrequently, Such as ‘03-0582’, ‘03-0441’, or ‘99-2495’

A backcross conversion of turfgrass plants of the present inventionhaving desirable looking turf when mowed infrequently, such as‘03-0582’, ‘03-0441’, or ‘99-2495’ occurs when DNA sequences areintroduced through backcrossing (Poehlman, Breeding Field Crops, p. 204(1987), with ‘03-0582’, ‘03-0441’, or ‘99-2495’ utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast two or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see Openshaw, S. J. et al., “Marker-assisted Selection inBackcross Breeding” In: Proceedings Symposium of the Analysis ofMolecular Data, August 1994, Crop Science Society of America, Corvallis,Oreg., where it is demonstrated that a backcross conversion can be madein as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes vsunlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (SeeHallauer et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. 1998). Desired traits that may be transferred through backcrossconversion include, but are not limited to, sterility (nuclear andcytoplasmic), fertility restoration, nutritional enhancements, droughttolerance, nitrogen utilization, altered fatty acid profile, alteredcarbohydrate profile, modified oil production, industrial enhancements,disease resistance (bacterial, fungal or viral), insect resistance andherbicide resistance. In addition, an introgression site itself, such asan FRT site, Lox site or other site-specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety. In someembodiments, the number of loci that may be backcrossed into ‘03-0582’,‘03-0441’, or ‘99-2495’ is at least 1, 2, 3, 4, or 5 and/or no more than6, 5, 4, 3, or 2. A single locus may contain several transgenes, such asa transgene for disease resistance that, in the same expression vector,also contains a transgene for herbicide resistance. The gene forherbicide resistance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of a site-specificintegration system allows for the integration of multiple genes at theconverted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant and easily recognized traits.

One process for adding or modifying a trait or locus in turfgrass plantsof the present invention having desirable looking turf when mowedinfrequently, such as bluegrass variety ‘03-0582’, ‘03-0441’, or‘99-2495’ comprises crossing ‘03-0582’, ‘03-0441’, or ‘99-2495’ plantsgrown from ‘03-0582’, ‘03-0441’, or ‘99-2495’ seed with plants ofanother bluegrass variety that comprise the desired trait or locus,selecting F₁ progeny plants that comprise the desired trait or locus toproduce selected F₁ progeny plants, crossing the selected progeny plantswith the ‘03-0582’, ‘03-0441’, or ‘99-2495’ plants to produce backcrossprogeny plants, selecting for backcross progeny plants that have thedesired trait or locus and the morphological characteristics ofbluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ to produce selectedbackcross progeny plants; and backcrossing to ‘03-0582’, ‘03-0441’, or‘99-2495’ three or more times in succession to produce selected fourthor higher backcross progeny plants that comprise said trait or locus.The modified ‘03-0582’, ‘03-0441’, or ‘99-2495’ may be furthercharacterized as having the physiological and morphologicalcharacteristics of bluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’and/or may be characterized by percent similarity or identity to‘03-0582’, ‘03-0441’, or ‘99-2495’ as determined by SSR markers. Theabove method may be utilized with fewer backcrosses in appropriatesituations, such as when the donor parent is highly related or markersare used in the selection step. Desired traits that may be used includethose nucleic acids known in the art, some of which are mentionedherein, that will affect traits through nucleic acid expression orinhibition. Desired loci include the introgression of FRT, Lox and othersites for site specific integration, which may also affect a desiredtrait if a functional nucleic acid is inserted at the integration site.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asturfgrass plants of the present invention having desirable looking turfwhen mowed infrequently, such as ‘03-0582’, ‘03-0441’, or ‘99-2495’ andanother bluegrass variety having one or more desirable characteristicsthat is lacking or which complements ‘03-0582’, ‘03-0441’, or ‘99-2495’.If the two original parents do not provide all the desiredcharacteristics, other sources can be included in the breedingpopulation. In the pedigree method, superior plants are selfed andselected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅, etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. In some embodiments, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent but at the same time retain manycomponents of the non-recurrent parent by stopping the backcrossing atan early stage and proceeding with selfing and selection. For example, aturfgrass variety may be crossed with another variety to produce a firstgeneration progeny plant. The first generation progeny plant may then bebackcrossed to one of its parent varieties to create a BC₁ or BC₂.Progeny are selfed and selected so that the newly developed variety hasmany of the attributes of the recurrent parent and yet several of thedesired attributes of the non-recurrent parent. This approach leveragesthe value and strengths of the recurrent parent for use in new turfgrassvarieties.

Therefore, an embodiment is a method of making a backcross conversion ofturfgrass plants of the present invention having desirable looking turfwhen mowed infrequently, such as bluegrass variety ‘03-0582’, ‘03-0441’,or ‘99-2495’, comprising the steps of crossing a plant of bluegrassvariety ‘03-0582’, ‘03-0441’, or ‘99-2495’ with a donor plant comprisinga desired trait, selecting an F₁ progeny plant comprising the desiredtrait, and backcrossing the selected F₁ progeny plant to a plant ofbluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’. This method mayfurther comprise the step of obtaining a molecular marker profile ofbluegrass variety ‘03-0582’, ‘03-0441’, or ‘99-2495’ and using themolecular marker profile to select for a progeny plant with the desiredtrait and the molecular marker profile of ‘03-0582’, ‘03-0441’, or‘99-2495’. In one embodiment the desired trait is a mutant gene ortransgene present in the donor parent.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny bluegrass seed byadding a step at the end of the process that comprises crossingturfgrass plants of the present invention having desirable looking turfwhen mowed infrequently, such as ‘03-0582’, ‘03-0441’, or ‘99-2495’ withthe introgressed trait or locus with a different bluegrass plant andharvesting the resultant first generation progeny bluegrass seed.

Transgenic Turfgrass

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, transgenic variants of the turfgrass varieties of thepresent invention may contain at least one transgene but could containat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or no more than 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Over the last fifteen to twentyyears several methods for producing transgenic plants have beendeveloped, and the present invention also relates to transgenic variantsof the claimed turfgrass varieties of the present invention.

Genetic engineering of the plants of the present invention includesvarious methods for crop improvement, including transgenic modification,xenogenic modification, intragenic modification, famigenic modificationand cisgenic modification.

One embodiment of the invention is a process for producing turfgrassvarieties further comprising a desired trait, said process comprisingtransforming a turfgrass plant with a transgene that confers a desiredtrait. Another embodiment is the product produced by this process. Inone embodiment the desired trait may be one or more of herbicideresistance, insect resistance, disease resistance or modified fatty acidor carbohydrate metabolism. In one embodiment the desired trait may beincreased or modified oil content. The specific gene may be any known inthe art or listed herein, including: a polynucleotide conferringresistance to imidazolinone, sulfonylurea, glyphosate, glufosinate,triazine, benzonitrile, cyclohexanedione, phenoxy proprionic acid andL-phosphinothricin; a polynucleotide encoding a Bacillus thuringiensispolypeptide, or a polynucleotide conferring resistance to one or morenematodes, Phytophthora root rot, or other fungi, or one or moreviruses.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109, 1999). In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

A genetic trait which has been engineered into the genome of aparticular turfgrass plant may then be moved into the genome of anotherturfgrass variety using traditional breeding techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachis commonly used to move a transgene from a transformed bluegrassvariety into an already developed bluegrass variety, and the resultingbackcross conversion plant would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes,coding sequences, inducible, constitutive, and tissue specificpromoters, enhancing sequences, and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. No. 6,118,055.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedturfgrass plants using transformation methods as described below toincorporate transgenes into the genetic material of the turfgrassplant(s).

With transgenic plants according to the methods and variety describedherein, a foreign or endogenous protein or oil can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign or endogenous protein or oil then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a one embodiment, the transgenic plant provided forcommercial production of foreign or endogenous protein or oil is aturfgrass plant. In another embodiment, the biomass of interest isleaves, stems, or other plant parts. For the relatively small number oftransgenic plants that show higher levels of expression, a genetic mapcan be generated, primarily via conventional RFLP, PCR and SSR analysis,which identifies the approximate chromosomal location of the integratedDNA molecule. For exemplary methodologies in this regard, see Glick andThompson, Methods in Plant Molecular Biology and Biotechnology, CRCPress, Boca Raton 269:284 (1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant.

Expression Vectors for Turfgrass Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol.Biol. 7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al.,EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. USA 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available (Molecular Probes publication2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991)). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie et al., Science 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Turfgrass Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific”. A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

The choice of regulatory regions to be included in a recombinantconstruct depends upon several factors, including, but not limited to,efficiency, selectability, inducibility, desired expression level, andcell- or tissue-preferential expression. It is a routine matter for oneof skill in the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. Transcription of a nucleic acid can be modulated ina similar manner. Some suitable regulatory regions initiatetranscription only, or predominantly, in certain cell types. Methods foridentifying and characterizing regulatory regions in plant genomic DNAare known, including, for example, those described in the followingreferences: Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al.,Plant Cell, 1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988);Meier et al., Plant Cell, 3:309-316 (1991); and Zhang et al., PlantPhysiology, 110:1069-1079 (1996). Examples of various regulatory regionsare described in more detail in U.S. Patent Application Ser. Nos.US20080072340, US20080044898, US20070277269, US20070226830,US20070136839, US 20070124834, and US 20060107346. It will beappreciated that a regulatory region may meet criteria for oneclassification based on its activity in one plant species, and yet meetcriteria for a different classification based on its activity in anotherplant species. Examples of regulatory regions include broadly expressingpromoters, root promoters, maturing endosperm promoters, ovary tissuepromoters, embryo sac/early endosperm promoters, embryo promoters,photosynthetic tissue promoters, vascular tissue promoters, induciblepromoters, basal promoters, or other regulatory regions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in turfgrass. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in perennialturfgrass. With an inducible promoter the rate of transcriptionincreases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571(1993)); In2 gene from maize which responds to benzenesulfonamideherbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991)and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressorfrom Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). Aparticularly preferred inducible promoter is a promoter that responds toan inducing agent to which plants do not normally respond. An exemplaryinducible promoter is the inducible promoter from a steroid hormonegene, the transcriptional activity of which is induced by aglucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci. USA88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in turfgrass or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in turfgrass.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXbal/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in turfgrass.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in turfgrass. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondrion or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20:49 (1992); Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Frontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are becoming available forthe bluegrass genome. Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of interest. Through thetransformation of turfgrass the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance andother traits, such as oil content. DNA sequences native to turfgrass aswell as non-native DNA sequences can be transformed into turfgrass andused to alter levels of native or non-native proteins. Variouspromoters, targeting sequences, enhancing sequences, and other DNAsequences can be inserted into the genome for the purpose of alteringthe expression of proteins. Reduction of the activity of specific genes(also known as gene silencing, or gene suppression) is desirable forseveral aspects of genetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Likewise, by means of the present invention, additional genes ofinterest can be expressed in transformed plants. Exemplary genesimplicated in this regard include, but are not limited to, thosecategorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example Jones et al., Science266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. tomato encodes a proteinkinase); Mindrinos et al. Cell 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell & Woffenden, (2003) TrendsBiotechnol. 21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82.

B. A gene conferring resistance to a pest, such as a nematode. See e.g.,PCT Application WO 96/30517; PCT Application WO 93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, Van Damme et al., Plant Molec. Biol.24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT application US93/06487 which teaches the use of avidin and avidin homologues aslarvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata); Chattopadhyay et al. (2004) CriticalReviews in Microbiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod67 (2): 300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11):1515-1539; Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; andVasconcelos & Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S.Pat. No. 5,266,317 to Tomalski et al., which discloses genes encodinginsect-specific, paralytic neurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 (Scott et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene, U.S. Pat. Nos. 7,145,060,7,087,810 and 6,563,020.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852, which disclose peptide derivatives oftachyplesin which inhibit fungal plant pathogens, and PCT application WO95/18855 and U.S. Pat. No. 5,607,914 which teaches syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virusand tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., Current Biology, 5(2)(1995); Pieterse & Van Loon (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich (2003) Cell 113(7):815-6.

U. Antifungal genes. See Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs et al., Planta 183:258-264 (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. Pat. No. 6,875,907.

V. Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. No. 5,792,931.

W. Cystatin and cysteine proteinase inhibitors. See U.S. Pat. No.7,205,453.

X. Defensin genes. See WO 03/000863 and U.S. Pat. No. 6,911,577.

Y. Genes that confer resistance to Phytophthora root rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexanediones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSPS which can confer glyphosateresistance. U.S. Pat. No. 5,627,061 to Barry et al. also describes genesencoding EPSPS enzymes. See also U.S. Pat. Nos. 6,566,587; 6,338,961;6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908;5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366;5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE37,287 E; and 5,491,288; and international publications EP1173580; WO01/66704; EP1173581 and EP1173582, which are incorporated herein byreference for this purpose. Glyphosate resistance is also imparted toplants that express a gene that encodes a glyphosate oxido-reductaseenzyme as described more fully in U.S. Pat. Nos. 5,776,760 and5,463,175, which are incorporated herein by reference for this purpose.In addition glyphosate resistance can be imparted to plants by the overexpression of genes encoding glyphosate N-acetyltransferase. See, forexample, U.S. application Ser. No. 10/427,692. A DNA molecule encoding amutant aroA gene can be obtained under ATCC accession number 39256, andthe nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a PAT gene is provided in European application No. 0 242 246to Leemans et al. DeGreef et al., Bio/Technology 7:61 (1989) describethe production of transgenic plants that express chimeric bar genescoding for phosphinothricin acetyl transferase activity. Exemplary ofgenes conferring resistance to phenoxy proprionic acids andcyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2,and Acc2-S3 genes described by Marshall et al., Theor. Appl. Genet.83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821,which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al., 1991; VickiChandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pinrecombinase of E. coli (Enomoto et al., 1983), and the R/RS system ofthe pSR1 plasmid (Araki et al., 1992).

6. Genes that Affect Abiotic Stress Resistance.

Genes that affect abiotic stress resistance (including but not limitedto flowering, pod and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705,U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No.6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat.No. 6,717,034, U.S. Pat. No. 6,801,104, WO 2000/060089, WO 2001/026459,WO 2001/035725, WO 2001/034726, WO 2001/035727, WO 2001/036444, WO2001/036597, WO 2001/036598, WO 2002/015675, WO 2002/017430, WO2002/077185, WO 2002/079403, WO 2003/013227, WO 2003/013228, WO2003/014327, WO 2004/031349, WO 2004/076638, WO 98/09521, and WO99/38977 describing genes, including CBF genes and transcription factorseffective in mitigating the negative effects of freezing, high salinity,and drought on plants, as well as conferring other positive effects onplant phenotype; US 2004/0148654 and WO 01/36596 where abscisic acid isaltered in plants resulting in improved plant phenotype such asincreased yield and/or increased tolerance to abiotic stress; WO2000/006341, WO 04/090143, U.S. application Ser. No. 10/817,483 and U.S.Pat. No. 6,992,237 where cytokinin expression is modified resulting inplants with increased stress tolerance, such as drought tolerance,and/or increased yield. Also see WO 02/02776, WO 2003/052063,JP2002281975, U.S. Pat. No. 6,084,153, WO 01/64898, U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness). For ethylene alteration, see US 20040128719,US 20030166197 and WO 2000/32761. For plant transcription factors ortranscriptional regulators of abiotic stress, see e.g. US 20040098764 orUS 20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g. WO97/49811 (LHY), WO 98/56918 (ESD4), WO 97/10339 and U.S. Pat No.6,573,430 (TFL), U.S. Pat No. 6713663 (FT), WO 96/14414 (CON), WO96/38560, WO 01/21822 (VRN1), WO 00/44918 (VRN2), WO 99/49064 (GI), WO00/46358 (FRI), WO 97/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No.6307126 (GAI), WO 99/09174 (D8 and Rht), and WO 2004/076638 and WO2004/031349 (transcription factors).

Methods for Turfgrass Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc. Boca Raton, 1993) pages67-88. In addition, expression vectors and in-vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation where DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987);Sanford, J. C., Trends Biotech. 6:299 (1988); Klein et al., Bio/Tech.6:559-563 (1988); Sanford, J. C. Physiol Plant 7:206 (1990); Klein etal., Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580(Christou, et al.), issued May 14, 1991 and U.S. Pat. No. 5,322,783(Tomes, et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985); Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described (Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994)).

Following transformation of bluegrass target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

Genetic Marker Profile through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Cregan et. al,

“An Integrated Genetic Linkage Map of the Soybean Genome” Crop Science39:1464-1490 (1999), and Berry et al., Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize InbredLines and Soybean Varieties” Genetics 165:331-342 (2003).

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR detection is done by useof two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties it is preferable if all SSRprofiles are performed in the same lab.

Tissue Culture

Further reproduction of the turfgrass varieties of the present inventioncan occur by tissue culture and regeneration. Tissue culture of varioustissues of turfgrass and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Bradley, D.E. et al. 2001. Effects of cultivar, explant treatment, and mediumsupplements on callus induction and plantlet regeneration in perennialbluegrass. Int. Turfgrass Soc. Res. J. 9:152-156; Cao, M. X., et al.2006. Transformation of recalcitrant turfgrass cultivars throughimprovement of tissue culture and selection regime. Plant, Cell, TissueOrgan Culture. 85:307-316; WenZhen, L. et al. Factors effecting ontissue culture of perennial ryegrass (Lolium perenne L.), Forest Res.2004. 17:95-101 Thus, another aspect of this invention is to providecells which upon growth and differentiation produce turfgrass plantshaving the physiological and morphological characteristics of theturfgrass plants of the present invention.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, culms, leaves,stems, roots, root tips, anthers, pistils and the like. Means forpreparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

EXAMPLES

The following examples are provided to further illustrate the presentinvention and are not intended to limit the invention beyond thelimitations set forth in the appended claims. The three cultivars listedbelow are the first in a series of new turfgrass varieties. Thefollowing descriptions characterize their breeding history, development,and the seed production characteristics of these cultivars.

Example 1 Method of Producing a Turfgrass Plant having Desirable LookingTurf when Mowed Infrequently

In the field hybridization nursery, individual spaced plants areidentified with promising characteristics such as the short growth ofleaves (defined as a preponderance of green leaf material in the 0 to 4inch zone above the soil surface), adequate seed heads (defined as 15 gor more of clean seed recovered from one individual spaced plant), andacceptable apomixes (defined as 80% or higher). These promising plantsare individually hand harvested and cleaned of chaff and seed areplanted in replicated turf trials, subject to mowing once every 4 weeksor less frequent. Each plot in the experiment is evaluated monthlyduring the growing season, using a visual rating scale of 1 to 9, where9 is highly desirable turf and 5 is minimal acceptable quality, and 1 istotally brown or dead. During one or more evaluation dates, thechlorophyll meter is used to evaluate plots to impartially differentiategreen tissue from lifeless brown tissue created from the scalpingprocess.

Most turfgrass managers practice the “one third rule” for clipping lawnsand golf courses. The “one third rule” states that a maximum of onethird of the leaf area should be removed during any one mowing.Deviating from this “one third rule” could lead to an unacceptablequantity of the plant's photosynthetic surface being removed. As aresult, the plant goes into a shock mode and sacrifices leaves inexchange for survival. Plant varieties that grow vertically very tall orextremely fast are more subject to damage from defoliation. The visualresult is that the plant rapidly turns brown within 24 hours and maytake days or weeks to recover. An additional result of this shock is theloss of shoot density of the plant, defined as the number of livevegetative shoots per square centimeter of ground surface. The presentinvention described herein deals with plants that do not exhibit thisbrowning shock when they are subject to infrequent mowing.

The present invention provides a method of producing a turfgrass planthaving desirable-looking turf when mowed infrequently, said methodcomprises selfing (pollinating a plant and hybridizing it with its ownpollen) for one or more generations to create a plant with short growthof leaves (defined as a preponderance of green leaf material in the 0 to4 inch zone above the soil surface), adequate seed heads (defined as 15g or more of clean seed recovered from one individual spaced plant), andacceptable apomixes (defined as 80% or higher). The hybridization cantake place in a confined greenhouse using pollen restricting bags, ortake place in the field surrounded by clones of the same genotype, orany combination of these two methods. The progeny of said hybridizationsare then field screened for approximately 15 months to identify plantswith characteristics different than the maternal parent line; suchdifferences are most commonly identified during the vegetative orpre-seedhead phase, but also may be identified during seedheadexpression, and include differences in color and width of the leavesprior to seedhead expression and a different date when the seedheademerges from the sheath of said turfgrass genotype. The method of thepresent invention unexpectedly produced turfgrass plants having verybasal growth habit, with leaves growing close to the ground rather thangrowing tall, which have desirable-looking turf when mowed infrequently.

Example 2 Development and Characteristics of Kentucky Bluegrass Plant‘03-0582’

‘03-0582’ Kentucky bluegrass, also known by the designation ‘J-0582’,originated as a low-growing, apomictic, single-plant selection from theprogeny of Jacklin Seed breeding line ‘98-2111’. Breeding line ‘98-2111’was self pollinated to produce ‘03-0582’. Breeding line ‘98-2111’originated as a selected low-growing progeny of breeding line ‘96-0305’,first identified and harvested in the field in May 1996. Breeding line‘96-0305’ had a medium seed yield potential, 50% apomixis, medium-earlyreproductive maturity, and an average culm length of 45 cm. Based on theprior art, it was totally unexpected that a variety with low apomixeswould give rise to a variety with acceptable or high levels of apomixes.Breeding line ‘96-0305’ was a selection from the progeny of a hybridcross between Absolute Kentucky bluegrass pollinated by BlueChip,created as a paired field cross.

Seed harvested from ‘03-0582’ was used to establish infrequently mowedtrials in Idaho in 2006, and in Maryland and Ohio in 2007. ‘03-0582’ wasselected for release based on its turf quality performance in all monthsof the year under twice yearly mowing, as well as its seed productioncharacteristics in Washington State.

‘03-0582’ is an ideal plant form for infrequent mowing with low foliageand an unexpected proliferation of large dense seed heads on shortstrawed culms. At anthesis the stand and panicles have a yellowish greenappearance with very little purplish coloration with the exception ofthe anthers. The panicles take on an almost whitish cast when seen froma distance; however, up close several of the florets and sometimesentire panicles have an appearance like they were dipped in diffusepurple dye. Spaced nursery plants are shaped like a cupcake withvertical, uniformly length straw and a proliferation of seed heads atthe top. There is a difference in culm length by about 50% from thecenter to the perimeter of the plant. The foliage of the space plants isdark green in color and broad in width. Heading is considered medium inmaturity however anthesis is occurring on the same day as with the later5-Steps Above type varieties (e.g., NuGlade and Award).

Culms are smooth to the touch in both directions and medium green incolor. Flag leaves are deep bluish green in color and relatively short.They are smooth to the touch in the upward direction and rough to thetouch when felt in the direction toward the base. The flag leaf nodejunction is vertical with very little to no bend and little to nocoloration difference from the culm itself. Panicles are small in sizewith a good number of florets that are medium to small in size. Thepanicles have numerous branches, more than some varieties and thebranches are mostly oriented in the horizontal to downward directionwith little to no waviness. The tips are mostly drooping, as are thesecond or third nodes from the tip; the rest are somewhat ascending.There is little to no influence of soil on seed head productivity excepton the area of the field that received little to no water.

Progeny trials were conducted in spaced-plant nurseries, establishedJune 2010, to determine the level of apomixis. A survey of 579 plants of‘03-0582’ showed that 4.2% of plants were variants in the vegetative(pre-flowering) stage, 1.7% were heading maturity variants, 0.3% wereseedhead variants, 0% were variants appearing in the dry-down phase, and2.5% were miniature plants. Many of the variants have shorter culms thanthe majority plant form with less purple panicle coloration. Variantsmay not be obvious in commercial seed production due to the maskingeffect of bulk populations. The spaced-plant apomixis rate of ‘03-0582’averages 91%, but it varies from year to year depending on growingconditions.

Variants in this variety appear primarily during the vegetative growthstage before heading. The majority of the variants have culm lengthsequal to or shorter than the majority plant form and as such will berelatively inconspicuous in seed production. Soil variations make thevariety appear non-uniform in places however this is an adjustment ofthe variety to soil and not a difference in genetics. Most of thevariants have a similar vegetative form to the majority with theexception of more vertical or less vertical culm orientation.Approximately 2.5% of plants are miniature plants which will not beapparent in seed production.

‘03-0582’ Kentucky bluegrass is a stable and uniform variety. Allseedlots evaluated have produced turf of comparable quality andacceptable uniformity. Aberrant progeny are rogued from Breeders,Foundation, and Registered fields to insure continued uniformity andstability, but they will continue to occur in every generation.

‘03-0582’ is a versatile Kentucky bluegrass variety, with applicationson golf courses, sod farms, sports fields, home lawns, roadsides,cemeteries, and other turf areas, where bluegrass is well adapted.‘03-0582’ performs well in full sun or partial shade.

Example 3 Development and Characteristics of Kentucky Bluegrass Plant‘03-0441’

‘03-0441’ Kentucky bluegrass, also known by the designation ‘J-0441’,originated as a low-growing, apomictic, single-plant selection from theprogeny of Jacklin Seed breeding line ‘01-0307’. Breeding line ‘01-0307’was self pollinated to produce ‘03-0441’. Breeding line ‘01-0307’originated as the low-growing progeny of a hybrid field cross of ChicagoII Kentucky bluegrass pollinated by breeding line ‘95-2986’. Breedingline ‘95-2986’ had an excellent record in a 1997 turf trial in Idaho anda medium record in a 1999 trial in Maryland. It was first identified andharvested in the field in May 1995. Breeding line ‘95-2986’ was aselection from the hybrid progeny of Midnight Kentucky bluegrasspollinated by Limousine, created as a paired field cross Jun. 3, 1991.

Seed harvested from ‘03-0441’ was used to establish infrequently mowedtrials in Idaho in 2006, and in Maryland and Ohio in 2007. ‘03-0441’ wasselected for release based on its turf quality performance in all monthsof the year under twice yearly mowing, as well as its seed productioncharacteristics in Washington State.

‘03-0441’ is a classic low-growing plant with medium reproductivematurity, stocky culms and a dense proliferation of seed heads,featuring unexpected excellent powdery mildew disease resistance. Spaceplants average 35 cm across after one year's growth from rhizomes. Colorof the stand at anthesis is light green with almost no purple colorationwhatsoever. Plants are strongly blocky in shape with little to nolodging towards the perimeter of space plants. There is little to noinfluence of soil from plant to plant on productivity.

Panicles are small in size, however florets are large and paniclenumbers are high. There is some tapering of culm length towards theperimeter of space plants giving the top of the space plant a roundedappearance. The center culms are approximately 10 cm taller than theculms at the perimeter of the plant. Culms are smooth to the touch inthe upward direction and very lightly rough in the downward direction.Panicles are drooping at the tip and florets are drooping across most ofthe panicle but with the two lower nodes ascending. There is no wavinessto the branching. Flag leaf margins are rough when felt in the downwarddirection and slightly rough in the upward direction. Flag leaf node isolive green in color and about 2 mm wide and slightly broader than theculm itself. Foliage color of space plants is medium dark green and flagleaves are distinctly bluish. Culms are medium green in color.

Progeny trials were conducted in spaced-plant nurseries, establishedJune 2010, to determine the level of apomixis. A survey of 974 plants ofshowed that 12.8% of plants were variants in the vegetative(pre-flowering) stage, 1.4% were heading maturity variants, 0.1% wereseedhead variants, 0.9% were variants appearing in the dry-down phase,and 2.1% were miniature plants. Many of the variants have shorter culmsthan the majority plant form with less purple panicle coloration.Variants may not be obvious in commercial seed production due to themasking effect of bulk populations. The spaced-plant apomixis rate of‘03-0441’ averages 83%, but it varies depending on growing conditions.

Variants are distributed across all phases of maturation. The primaryvariant has a more yellow panicle and lighter colored foliage than themajority plant form. About 1% of variants show a strongly drooping seedhead with a longer panicle and lighter foliage. Most variants seem toshare the stocky upright growth habit of the majority plant form.

‘03-0441’ Kentucky bluegrass is a stable and uniform variety. Allseedlots evaluated have produced turf of comparable quality andacceptable uniformity. Aberrant progeny are rogued from Breeders,Foundation, and Registered fields to insure continued uniformity andstability, but they will continue to occur in every generation.

‘03-0441’ is a versatile Kentucky bluegrass variety, with applicationson golf courses, sod farms, sports fields, home lawns, roadsides,cemeteries, and other turf areas, where bluegrass is well adapted.‘03-0441’ performs well in full sun or partial shade.

Example 4 Development and Characteristics of Kentucky Bluegrass Plant‘99-2495’

‘99-2495’ Kentucky bluegrass, also known by the designation ‘J-2495’,originated as a low-growing, apomictic, single-plant selection from theprogeny of Jacklin Seed breeding line ‘97-0429’. Breeding line ‘97-0429’was self pollinated to produce ‘99-2495’. Breeding line ‘97-0429’ had amedium leaf color and reproductive maturity, a culm length of 54 cm, ahigh level of apomixis, and was susceptible to ergot and powdery mildew.Breeding line ‘97-0429’ originated as a low-growing progeny of a hybridcross of BlueChip Kentucky bluegrass pollinated by Blacksburg, createdas a paired field cross Jun. 3, 1999.

Seed harvested from ‘99-2495’ was used to establish infrequently mowedtrials in Idaho in 2006, and in Maryland and Ohio in 2007. ‘99-2495’ wasselected for release based on its turf quality performance in all monthsof the year under twice yearly mowing, as well as its seed productioncharacteristics in Washington State.

‘99-2495’ is a late maturing, shorter growing, fine textured version ofa standard 5-Steps Above variety. ‘99-2495’ has susceptibility topowdery mildew. Space plants average 45 cm across after one year'sgrowth from rhizomes. The most unexpected and distinctive characteristicof this variety is its extremely fine, extremely dark green foliage asspaced plants. Of all the varieties in the Breeder blocks, this one hasthe most fine, dark leaves. Seed head productivity is excellent withsome influence of plant-to-plant yield variation due to soil.

Panicle color at anthesis is light green with a diffusion of purplishcolor on the tips of florets. However, it does not appear as a distinctspeckling as it does in some cultivars. Culms are medium green in colorand are mostly smooth with only a bit of roughness. Flag leafs aresmooth when felt in the upward direction and rough in the downwarddirection. Attachment node of flag leaves is the same color as the culmwith only a slight knob-like appearance, with little to no bend at thenode. Spaced plant form is mostly blocky with a uniform culm length withlittle tapering (maybe 5 cm) towards the perimeter of the plant.Panicles are medium in size and florets are medium in size. Internodelength gets shorter towards the tip of the panicle. There is a minoramount of waviness to the branching in the panicle, mostly towards thetip. Tips are mostly upright to slightly nodding. Most panicle branchingis ascending however the lowest node is horizontal to descending. Theattachment of the florets is horizontal to slightly downward.

Progeny trials were conducted in spaced-plant nurseries, establishedJune 2010, to determine the level of apomixis. A survey of 1044 plantsof ‘99-2495’ showed that 11.7% of plants were variants in the vegetative(pre-flowering) stage, 1.8% were heading maturity variants, 0.2% wereseedhead variants, 0.3% were variants appearing in the dry-down phase,and 0.8% were miniature plants. Many of the variants have shorter culmsthan the majority plant form with less purple panicle coloration.Variants may not be obvious in commercial seed production due to themasking effect of bulk populations. The spaced-plant apomixis rate of‘99-2495’ averages 85%, but it varies depending on growing conditions.

Variants in this cultivar appear during outbreaks of powdery mildewwhich shows resistant types. Most of the vegetative variants areslightly taller than the majority plant form. About 0.2% of plants arean open, easily lodged plant with drooping seed heads. The headingmaturity variants differ from the majority plant form by a slightlytaller and more yellow panicles.

‘99-2495’ Kentucky bluegrass is a stable and uniform variety. Allseedlots evaluated have produced turf of comparable quality andacceptable uniformity. Aberrant progeny are rogued from Breeders,Foundation, and Registered fields to insure continued uniformity andstability, but they will continue to occur in every generation.

‘99-2495’ is a versatile Kentucky bluegrass variety, with applicationson golf courses, sod farms, sports fields, home lawns, roadsides,cemeteries, and other turf areas, where bluegrass is well adapted.‘99-2495’ performs well in full sun or partial shade.

Example 5 Physiological and Morphological Characteristics of NewKentucky Bluegrass Varieties

Table 1 shows a summary of the data obtained from trials held in 2010.The most promising varieties were planted into a spaced plant breederblocks in Connell, Washington, in the heart of Kentucky bluegrass seedproduction country. The goal was to see how they performed in seedproduction, to pick the best of the best. After a year of observationand data collection, the three best entries were chosen; they appear atthe top of Table 1. The remaining varieties in the table were not chosendue to one or more undesirable traits.

In Table 1 below, apomixis is the percentage of apomictic seedreproduction in the varieties. A value of 100 would indicate alloffspring are identical genetically. Data in the remaining columns werevisually rated on a 1 to 9 scale, with 9 equal to most low-growingcharacteristics, greatest powdery mildew resistance, highest seed yield,least soil variability from one plant to the next, and highest generalturfgrass quality ratings in Ohio and Maryland averaged across monthlyreadings taken over three growing seasons. Mean values over 5 would beconsidered good and values of 8 or more could be considered outstanding.Commercial standards (i.e. varieties that are being sold on the markettoday) were tested in the initial Idaho studies. All commercialstandards performed poorly compared to the promising experimentals ofthe present invention.

TABLE 1 Dwarf Dwarf Mildew Yield Soil var 2007 OH 2007 OH 2007 MD 2007MD ID 28apr11 16jun11 28apr11 16jun11 9 = none Rating 2008 Rating 2009Rating 2008 Rating 2009 Apomixis 03-0582 8 7 8 8 8 6.00 5.21 6.00 5.2191 03-0441 8 6.5 9 7 6 6.00 6.57 6.67 4.00 83 99-2495 5.5 5.5 6 5.5 65.67 5.98 6.33 6.17 85 02-2139 6 7 2 4 5 6.33 6.33 6.67 4.50 91 02-22176 5 7 5 3 5.00 4.76 7.33 5.71 89 93-1436 6 3 7 8 8 4.33 6.50 7.33 5.1787 93-1897 4 6 4 5 6 6.33 6.86 5.00 4.33 93 97-0428 6 6.5 4.5 6.5 5.55.00 6.55 6.00 5.50 91 99-2304 4 4 5 6 4 6.67 6.83 90 99-2891 6 7 7 6 74.67 5.61 6.67 5.67 95

As shown in Table 1, Kentucky bluegrass varieties designated ‘03-0582’,‘03-0441’, and ‘99-2495’ have the necessary attributes to provide abeautiful lawn with substantially reduced mowing requirements.

Example 6 Chlorophyll Concentration of new Kentucky Bluegrass Varieties

A Field Scout CM 1000™ chlorophyll meter was used for documenting thechlorophyll in field plots of infrequently mowed turf. In August 2009,1000+ experimental plots were planted in Post Falls, Id. These plotswere maintained under three-times-per-year mowing at 2.5-inch cut. Someplots did well under this mowing regime but most did not. A typical plotin the latter contained a majority of brown, lifeless stems and verylittle green leafy material. The chlorophyll meter picked up thesedifferences in the good and bad plots and put a number to it.

This procedure is different than the lab procedure described elsewherein that the CM 1000™ was detecting differences between the brownlifeless material and green leafy material in the plots maintained underinfrequent mowing. In the lab procedure, only green tissue was used forchlorophyll concentration analysis. Moreover, the CM 1000™ did notproduce chlorophyll readings in terms of parts per million ofchlorophyll in leaf tissue. Instead, it produced a reading of 0 to 999based on what the sensors detected in certain narrow wave bands(described below) which are sensitive to the spectral nature of thechlorophyll molecule. In the test plots, readings fell between 150 and600 on that scale.

Visual turfgrass quality ratings and the CM 1000™ chlorophyll scans werereconciled as shown in FIG. 1. Forty-two readings were taken on theplots described above on Oct. 29, 2012 using both visual rating and CM1000™ meter readings. These observations were graphed and were analyzedusing linear regression. Solving the regression equation for a turfquality rating of ‘5’ (the minimum acceptable number used in plantbreeding to develop the cultivars) the equivalent CM 1000™ reading wouldbe 341.7. The turfgrass plants of the present invention have a CM 1000™meter reading of 341.7 and above on turfgrass mowed once every 4 weeksor fewer, combined with a previously specified chlorophyll concentrationin green tissue—in other words, the ability to produce acceptable lawnturf while being mowed infrequently.

Regression analysis was performed to correlate the visual turfgrassquality ratings with Field Scout CM 1000™ scan results for test plotsmowed three times per year in Idaho and the results are shown in FIG. 1.Regression analysis indicated that a visual quality estimate of 5corresponds to a CM 1000™ meter reading of 341.7, with an unexpectedlyhigh correlation co-efficient of 0.86, and an R² value of 0.73, whichwas significant at the 0.0001 level. In FIG. 1, circles representindividual observations, a solid line indicates regression fit to thedata, and dashed lines indicate 95% confidence intervals.

Table 2 shows the chlorophyll concentration of Kentucky bluegrassvarieties designated ‘03-0582’, ‘03-0441’, and ‘99-2495’ compared tosimilar varieties; varieties that are under development are listed withtheir experimental numbers, while commercial varieties are shown withtheir cultivar names. Clippings were weighed (0.1g) and placed in aglass test tube (1.0 cm in width and 14.8 cm in length) with 10 mL ofdimethyl sulfoxide (DMSO) (Hiscox and Israelstam, 1979). Samples wereincubated in 65° C. water for 1.5 hr. Upon completion, samples werepassed through filter paper (Whatman 41, Whatman, England) and remainingextract (1 mL) transferred into cuvettes. Absorbance values wererecorded at 663 nm and 645 nm wavelengths using a Spectrophotometer.Blanks were initially run and also after every sixth sample. Thefollowing formula was used to calculate total shoot chlorophyll: (mgg⁻¹)=(8.02*D663+20.2 * D645)*0.1 (Amon, 1949). Column 1 shows the samplename and column 2 shows the total shoot chlorophyll in mg/g.

TABLE 2 Sample Total Shoot Chlorophyll (mg/g) 03-0582 2.57 99-2495 1.9103-0441 3.21 O2-2139 2.22 O2-2217 2.61 93-1897 2.02 99-2304 2.42 97-04282.82 99-2891 3.46 J-1770 2.35 Troy 1.18 Camas 1.40 Nublue 1.75 Merit1.23 Action 0.89 Thermal 1.54

As shown in Table 2, bluegrass varieties designated ‘03-0582’,‘03-0441’, and ‘99-2495’ all have chlorophyll concentrations above 1.8mg/g, whereas the commercial varieties Troy, Camas, Nublue, Merit,Action and Thermal all have chlorophyll concentrations below 1.8 mg/g.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

Deposit Information

A deposit of the bluegrass varieties ‘03-0582’, ‘03-0441’ and ‘99-2495’developed by the method of the present invention disclosed above andrecited in the appended claims has been made with the NationalCollections of Industrial, Food and Marine Bacteria (NCIMB), 23 StMachar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom. The date ofdeposit was Feb. 12, 2013. The deposit of 2,500 seeds was taken from thesame deposit maintained by Jacklin Seed since prior to the filing dateof this application. Upon allowance of any claims in this application,all restrictions on the availability to the public of the variety willbe irrevocably removed by affording access to a deposit of at least2,500 seeds of the same variety with the National Collections ofIndustrial, Food and Marine Bacteria (NCIMB), Aberdeen, Scotland, andthe deposit is intended to meet all of the requirements of 37 C.F.R.1.801-1.809. The NCIMB numbers are 42111, 42110, and 42112,respectively. The deposit will be maintained in the depository for aperiod of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedas necessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A method of producing a turfgrass plant having desirable looking turfwhen mowed infrequently, said method comprising: a. Planting turfgrassgenotypes; b. Selfing said turfgrass genotypes for one or moregenerations to produce progeny plants; c. Selecting progeny plantshaving short, basal growth of leaves, adequate production of seed heads,and an acceptable level of apomixis; d. Further selecting said progenyplants to identify plants with one or more characteristics differentfrom the maternal parental line of said turfgrass genotype and havingdesirable looking turf when mowed infrequently; and e. Harvesting seedor vegetative propagating material from said selected progeny plants. 2.A turfgrass plant produced by the method of claim
 1. 3. A turfgrassplant having desirable looking turf when mowed infrequently, whereinsaid turfgrass plant has a green leaf chlorophyll concentration above1.8 mg/g.
 4. A method of producing a bluegrass plant having desirablelooking turf when mowed infrequently, said method comprising: a.Planting bluegrass genotypes; b. Selfing said bluegrass genotypes forone or more generations to produce progeny plants; c. Selecting progenyplants having short, basal growth of leaves, adequate production of seedheads, and an acceptable level of apomixis; d. Further selecting saidprogeny plants to identify plants with one or more characteristicsdifferent from the maternal parental line of said bluegrass genotype andhaving desirable looking turf when mowed infrequently; and e. Harvestingseed or vegetative propagating material from said selected progenyplants.
 5. A bluegrass plant produced by the method of claim
 4. 6. Abluegrass plant having desirable looking turf when mowed infrequently,wherein said bluegrass plant has a green leaf chlorophyll concentrationabove 1.8 mg/g.
 7. The bluegrass plant of claim 6, wherein saidbluegrass plant has a field CM-1000 chlorophyll meter reading of between341.7 and 620.0.
 8. A grass seed produced by the plant of claim 6,wherein representative samples of seed of said grass were depositedunder NCIMB Nos. 42110, 42111, and
 42112. 9. Sod, comprising the grassplant of claim
 6. 10. The grass plant of claim 6, wherein said grass isplanted in a lawn.
 11. A vegetative sprig or clone of the grass plant ofclaim
 6. 12. A method for producing a bluegrass seed, comprisingcrossing two bluegrass plants and harvesting the resultant bluegrassseed, wherein at least one bluegrass plant is the bluegrass plant ofclaim
 6. 13. A bluegrass seed produced by the method of claim
 12. 14. Abluegrass plant, or a part thereof, produced by growing said seed ofclaim
 13. 15. The method of claim 12, wherein at least one of saidbluegrass plants is transgenic.
 16. A method of producing an herbicideresistant bluegrass plant, wherein said method comprises introducing agene conferring herbicide resistance into the plant of claim
 6. 17. Aherbicide resistant bluegrass plant produced by the method of claim 16,wherein the gene confers resistance to a herbicide selected from thegroup consisting of glyphosate, sulfonylurea, imidazolinone, dicamba,glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone,cyclohexanedione, triazine, and benzonitrile.
 18. A method of producinga pest or insect resistant bluegrass plant, wherein said methodcomprises introducing a gene conferring pest or insect resistance intothe bluegrass plant of claim
 6. 19. A pest or insect resistant bluegrassplant produced by the method of claim
 18. 20. The bluegrass plant ofclaim 19, wherein the gene encodes a Bacillus thuringiensis (Bt)endotoxin.
 21. A method of producing a disease resistant bluegrassplant, wherein said method comprises introducing a gene which confersdisease resistance into the bluegrass plant of claim
 6. 22. A diseaseresistant bluegrass plant produced by the method of claim
 21. 23. Amethod of introducing a desired trait into the plant of claim 6, whereinthe method comprises: a. crossing the plant of claim 6 with a plantselected from the group consisting of another bluegrass variety, aspecies of Poa, and another plant genus that comprises a desired traitto produce progeny plants; b. selecting one or more progeny plants thathave the desired trait to produce selected progeny plants; c.backcrossing the selected progeny plants with a plant of claim 6 toproduce backcross progeny plants; d. selecting for backcross progenyplants that have the desired trait; and e. repeating steps c. and d. twoor more times in succession to produce selected third or higherbackcross progeny plants that comprise the desired trait.
 24. The methodof claim 23, wherein the desired trait is a phenotypic trait, a gene, ora molecular marker.
 25. A bluegrass plant produced by the method ofclaim 24, wherein the plant has the desired trait and otherwise all thephysiological and morphological characteristics of the bluegrass plantof claim
 6. 26. A method of producing a commodity plant product, saidmethod comprising obtaining the plant of claim 6, or a part thereof, andproducing the commodity plant product from said plant or plant partthereof, wherein said commodity plant product is selected from the groupconsisting of protein concentrate, protein isolate and oil.
 27. Abluegrass variety having desirable looking turf when mowed infrequentlyselected from the group consisting of bluegrass varieties ‘03-0582’,‘03-0441’ and ‘99-2495’, wherein representative samples of seed of saidvarieties were deposited under NCIMB Nos. 42111, 42110, and 42112,respectively.